title: Pilocapine alters NMDA receptor expression and function in hippocampal neurons: NADPH oxidase and ERK1/2 mechanisms.
abstract: The molecular basis for epileptogenesis remains poorly defined, but repeated or prolonged seizures can cause altered hippocampal N-methyl D-aspartate receptor (NMDAR) stoichiometry, loss of hippocampal neurons, and aberrant mossy fiber sprouting. Using the muscarinic receptor 1 (m1R) agonist, pilocarpine (PILO), in hippocampal cell cultures we explored the early sequence of molecular events that occur within 24h of the initial insult and result in altered neuronal function during epileptogenesis. Our findings show that PILO-induced, m1R-mediated, inositol 1,4,5-trisphosphate (IP3) synthesis constitutes an early, crucial biochemical event required for NMDAR hyperactivation and subsequent NADPH oxidase (NOX) activation and NMDAR-independent ERK1/2 phoshorylation. Together, but not separately, NOX activation and ERK1/2 phosphorylation induce alterations in NMDAR stoichiometry through the upregulation of NR1 and NR2B subunits. Lastly, we demonstrated that PILO-mediated oxidative stress alters NMDAR function through the redox modulation of cysteine residues. The in vitro results related to thiol oxidation, NOX activation, ERK1/2 phosphorylation and NMDAR upregulation were confirmed in vivo, 24h after treatment of adult rats with PILO. These results obtained in PILO-treated primary hippocampal neurons--and confirmed in vivo at the same time-point after PILO--provide a better understanding of the early cellular responses during epileptogenesis and identify potential therapeutic targets to prevent development of chronic epilepsy.
authors.affiliation: Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA. rdimaio@hs.pitt.edu
authors.forename: Roberto
authors.initials: R
authors.lastname: Di Maio
authors.forename: Pier G
authors.initials: PG
authors.lastname: Mastroberardino
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Hu
authors.forename: Laura
authors.initials: L
authors.lastname: Montero
authors.forename: J Timothy
authors.initials: JT
authors.lastname: Greenamyre
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation / Q000187:drug effects; D010862:Pilocarpine / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D013226:Status Epilepticus / Q000139:chemically induced / Q000378:metabolism
chemical_list: D018721:Muscarinic Agonists; D016194:Receptors, N-Methyl-D-Aspartate; D010862:Pilocarpine; D019255:NADPH Oxidases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3